Synthesis and reactivity of 5-fluorouracil/cytarabine mutual prodrugs

Article abstract of DOI:10.1021/jo971076p

Two mutual prodrugs, in which two different anti-cancer drugs are attached to the same molecule via labile linkages, are synthesized and examined kinetically. One of the mutual prodrugs loses a drug component under physiological conditions within an hour, but the other mutual prodrug (having a longer spacer between the two drugs) is stable to chemical degradation even at hither pH values. Thus, enzymatic hydrolysis alone will release the two anti-cancer drugs. The potential value of anti-cancer mutual prodrugs is discussed.

Full text of DOI:10.1021/jo971076p

J . Org. Chem. 1997, 62, 9083-9088
9083
Syn th esis a n d Rea ctivity of 5-F lu or ou r a cil/Cyta r a bin e Mu tu a l
P r od r u gs
Fredric M. Menger* and Michael J . Rourk
Department of Chemistry, Emory University, Atlanta, Georgia 30322
Received J une 16, 1997X
Two mutual prodrugs, in which two different anti-cancer drugs are attached to the same molecule
via labile linkages, are synthesized and examined kinetically. One of the mutual prodrugs loses a
drug component under physiological conditions within an hour, but the other mutual prodrug (having
a longer spacer between the two drugs) is stable to chemical degradation even at higher pH values.
Thus, enzymatic hydrolysis alone will release the two anti-cancer drugs. The potential value of
anti-cancer mutual prodrugs is discussed.
In tr od u ction
action with different efficiencies. When it is desirable
to have the two drugs reach a site simultaneously, the
mutual prodrug strategy may be used to advantage
provided: “(a) the mutual prodrug is well absorbed; (b)
both components are released concomitantly and quan-
titatively after absorption; (c) the maximal effect of the
combination of the two drugs occurs at a 1:1 ratio; and
(d) the distribution and elimination of the two compo-
nents are similar”.4 A review of the subject appeared in
1994.5 The concept of a mutual prodrug is, clearly, well
established.
During the course of his studies on the selective toxicity
of chemotherapeutic agents in the late 1950s, Professor
Adrien Albert, an Australian medicinal chemist, wrote
the following: “Sometimes the substance, as adminis-
tered, is only a ‘prodrug’ which has to be broken down to
give the true drug. Examples of this kind are phenacetin,
chloral hydrate, pentavalent arsenicals, and two of the
antimalarials; pamaquin and proguanil. But this seems
to be a rare phenomenon, and it appears that most
substances act in the form in which they are given”.1
Albert’s prodrug concept was later deliberately incorpo-
rated into drug design, and prodrugs are now com-
monplace.
A prodrug is defined as a pharmacologically inactive
compound that is converted into an active drug by a
metabolic biotransformation.2 For example, a drug bear-
ing a hydroxyl group might be deactivated by acetylation.
After administration to a patient, the ester group is
hydrolyzed in vivo either by spontaneous chemical hy-
drolysis or by the action of esterases which are prevalent
in cells (eq 1). In this manner the drug is regenerated.
The work described herein involves a mutual prodrug
composed of two different anti-cancer drugs. A cancer
that is not totally eliminated by administration of a single
drug will, sooner or later, become resistant to that drug.
Administration of two drugs enhances the chance of
successful therapy because the likelihood of resistance
developing to two drugs is much less than to a single
drug.6 This has given rise to a large number of thera-
peutic regimes involving drug combinations. Two drugs
can be administered either sequentially or simulta-
neously. In a sequential treatment, there still exists a
possibility that cancer cells become resistant to the first
drug prior to being exposed to the second. This popula-
tion of resistant cells, albeit reduced in size from the
original burden, can in turn become resistant to the
second drug as well. Considerable guesswork is involved
in the timing with which a patient receives the two drugs
so as to minimize the chance of double-resistance.
drug-OH f drug-OAc in vivo8 drug-OH
(1)
prodrug
(inactive)
Enzymatic activation is preferred over chemical activa-
tion because the latter often involves inherently unstable
compounds. Prodrugs can be used to overcome many
problems including poor solubility, absorption, and pa-
tient acceptability as well as instability and toxicity.
Prodrugs can also lead to prolonged release. Often
prodrugs are classified into two types, namely those with
a removable attachment (“carrier-linked”) and those that
are “bioprecursors”, which are metabolically modified into
active compounds.3 The work herein concerns only the
former type.
A mutual prodrug consists of two different synergistic
drugs joined together. The two drugs may be connected
directly or by means of a linker. Ordinarily, when two
synergistic drugs are administered individually but
simultaneously, they will be transported to the site of
Alternatively, the two drugs can be given simulta-
neously. Although this would seem to reduce the prob-
ability of double-resistance, there are also problems with
the approach. Toxicity is, for example, a concern. If
dosages are reduced to compensate for toxicity, then this
could defeat the benefit of simultaneous administration.
Even if dosages remain unaltered, one cannot be certain
that simultaneous drug administration will kill 100% of
the cells. A cell in the center of a poorly vascularized
solid tumor might, for example, have a low probability
of receiving one “hit”, much less two. A resistance would
develop, therefore, to a single drug prior to a cell’s
exposure to a lethal dose of the second. In effect, the
tumor cells impose a time lag despite the simultaneous
X Abstract published in Advance ACS Abstracts, December 1, 1997.
(1) Albert, A. Nature 1958, 182, 421-423.
(2) Silverman, Richard B. The Organic Chemistry of Drug Design
and Drug Action; Academic Press: San Diego, 1992; p 352.
(3) Silverman, Richard B. The Organic Chemistry of Drug Design
and Drug Action; Academic Press: San Diego, 1992; pp 352-355.
(4) Silverman, Richard B. The Organic Chemistry of Drug Design
and Drug Action; Academic Press: San Diego, 1992; p 377.
(5) Singh, G.; Dev Sharma, P. Indian J . Pharm. Sci. 1993, 56(3),
69-79.
(6) Marx, J . L. Science 1986, 234, 818-820.
S0022-3263(97)01076-1 CCC: $14.00 © 1997 American Chemical Society

9084 J . Org. Chem., Vol. 62, No. 26, 1997
Menger and Rourk
bulk administration of two different drugs. An enhanced
chance of double-resistance by the cancer could also arise
if there were a cross-resistance as would occur when a
hit by one drug impairs the entry of the second into the
cell.
methylene group known, from the work of others,8 to be
readily removable (eq 2).
Now consider a mutual prodrug. Entry of a mutual
prodrug into a cancer cell necessarily exposes the cell to
the ravishes of two drugs simultaneously. Double-
resistance would be unlikely unless a lethal dose requires
the entry of multiple drug molecules that can occur,
under the given dosages, only over the course of a long
time period. Note that the cytotoxicity of a mutual
prodrug might exceed that provided by the corresponding
two drugs administered sequentially or simultaneously.
Thus, it is conceivable that a cancer cell is killed by a
dual hit from a mutual prodrug while surviving a hit from
one drug and then a hit by a second drug, say, 10 h later.
By way of analogy, a person might succomb to a simul-
taneous attack of malaria and flu while surviving the two
diseases when spaced 10 months apart.
Syn th esis
The synthesis of 1a ,b is shown in Scheme 1. Several
factors made the synthesis far more difficult than is
apparent in the figures: (a) Attachment of the 5-FU
moiety in 5a ,b created labile N-O ketal derivatives.
These functionalities were sensitive to base (which would
saponify the ester) and even to warm alcohol which can
destroy the functionalities via transesterification. (b) All
reactions subsequent to 5a ,b necessitated mild conditions
and chemoselective reagents owing to the delicacy of the
multifunctional compounds. Thus, use of heat, strong
oxidizing or reducing agents, and harsh reagents (such
as thionyl chloride) caused either yield-reducing side
reactions or outright decomposition. This limitation
greatly reduced the pool of usable reagents. (c) The
polarity imparted by both the multiple heteroatoms
required the use of highly polar solvents (DMF, DMSO,
and glacial acetic acid) for the later reactions. To counter
this problem, the synthetic intermediates were, with each
step, deliberately imparted with alternating polarity
(compare, for example, 4 and 5, 5 and 6, 6 and 7, and 7
and 8). This greatly facilitated purification by standard
flash chromatography. Despite these problems, relatively
simple syntheses were developed. Thus, with the excep-
tion of the first two steps in either synthesis, all trans-
formations were carried out simply by stirring the
reactants at room temperature. Yields ranged from
moderate to excellent.
Formation of the desired phenylsulfanyl methyl ester
3a ,b proceeded well, and treatment of this compound
with sulfuryl chloride, SO2Cl2, generated the desired
chloromethyl ester (4a ,b) plus the benzenesulfenyl chlo-
ride byproduct (8). Since 8 is highly reactive, it could
not be carried through and had to be removed. This is
normally accomplished by trapping with cyclohexene, to
which 8 rapidly adds. The products are then normally
separated by vacuum distillation. In our case, however,
product 4a ,b and the cyclohexene addition product boiled
at similar temperatures, so that separation by vacuum
distillation was not possible. In order to circumvent this
difficulty, we used higher boiling trapping agents for the
benzenesulfenyl chloride (including trans-stilbene and
squalene). This approach would have succeeded were it
not for the fact that product yields suffered from thermal
degradation during the high temperatures required for
vacuum distillation of 4a ,b. A milder method of purifica-
tion was needed.
The simplistic nature of the above analysis should be
appreciated. In actual fact, the lethality of drug therapy
depends on numerous factors which are, for the most
part, unknown: (a) the number of molecules per dose;
(b) the number of cells per tumor; (c) the percent of
utilizable drug molecules that survive metabolism, excre-
tion, long-term storage, and entry into healthy cells; (d)
the number of drug molecules required within a cancer
cell to kill it; (e) the rate of resistance development. This
is a complicated business best handled by making
reasonable assumptions and by applying Poisson statis-
tics to assess the percentage of cells receiving a lethal
hit (an exercise in which we are now engaged). For the
moment, however, it suffices to presume, without a firm
basis, that anti-cancer mutual prodrugs are worthwhile
candidates for study.
Two potential anti-cancer mutual prodrugs (1a , n )
2; 1b, n ) 4) synthesized in our laboratory differ only in
the length of the spacer. They are seen to possess two
interconnected cytotoxic drugs, 5-fluorouracil (5-FU) and
cytarabine (ara-C). 5-FU has been widely used with solid
tumors including breast cancer, whereas ara-C has been
used mainly in the treatment of acute leukemia and
lymphomas. The cytarabine moiety is linked to the
double prodrug through a hydrolyzable amide group. It
is known, incidentally, that attaching an acyl group to
the ara-C primary amine group decreases the drug’s
susceptibility to deactivation by cytidine deaminase;7 the
amide group therefore serves two functions. The 5-FU
moiety is attached to the double prodrug via an (acyloxy)-
We reasoned that if the trapping agent for 8 contained
a double bond plus a water-solublizing group, then the
addition product could be removed by an aqueous wash.
A number of such trapping agents were tested (including
trans-cinnamic acid and 3-cyclohexenecarboxylic acid),
(8) (a) Ozaki, S.; Watanabe, Y.; Hoshiko, T.; Mizuno, H.; Ishikawa,
K.; Mori, H. Chem. Pharm. Bull. 1984, 32, 733-738. (b) Mollgaard,
B.; Hoelgaard, A.; Bundgaard, H. Int. J . Pharm. 1982, 12, 153-162.
(c) J ohansen, M.; Bundgaard, H.; Falch, E. Int. J . Pharm. 1983 13,
89-98.
(7) Tsuro, T.; Iida, H.; Hori, K.; Tsukagoshi, S.; Sakurai, Y. Cancer
Res. 1981, 41, 4484-4488.

5-Fluorouracil/Cytarabine Mutual Prodrugs
J . Org. Chem., Vol. 62, No. 26, 1997 9085
Sch em e 1a
but cis-4-cyclohexene-1,2-dicarboxylic acid (9) was found
to be by far the most useful (eq 3). This procedure is
recommended as an attractive variation on the published
theme.9
revert to the active drug prematurely. It became neces-
sary, therefore, to examine the hydrolysis kinetics of 1a
and 1b. Note that these mutual prodrugs possess two
labile groups connecting the drugs to the spacer. The
questions thus arise as to which of the two drugs cleaves
off first and how soon thereafter the second drug is freed.
In order to answer such questions of reactivity, we first
studied models of the mutual prodrugs in which each side
could be examined without interference from the other.
With regard to the reactivity of the 5FU portion of the
mutual prodrugs, mention should be made of the work
from the Bundgaard group8b,c who measured the reactiv-
ity of similar 5-FU prodrugs. It was found that the
Attachment of 5-FU to 4a ,b in Scheme 1 according to
a literature procedure8a proceeded as described. Only one
product, 5a ,b, was obtained; there was no sign of the N3
alkylation product. Catalytic transfer hydrogenation
(CTH) debenzylation of 5a ,b in glacial acetic acid, using
1,4-cyclohexadiene as the hydrogen source,10 gave 6a ,b
in high yield. Esterification of 6a ,b with pentafluorophe-
nol using a water-soluble carbodiimide, 1-(3-(dimethy-
lamino)propyl)-3-ethylcarbodiimide hydrochloride (EDCI‚-
HCl), gave activated ester 7a ,b. Immediate reaction of
7a ,b with free-base ara-C in DMF afforded the desired
1a ,b.11
observed rates of hydrolysis of N
1- or N3-(hydroxylmethyl)
5-FU in the pH range 2-10 are directly proportional to
the hydroxide ion activity. Thus plots of log kobs vs pH
were linear with a slope of 1.0 which is consistent with
the mechanism in eq 4. Bundgaard used two methods
Kin etics
Chemical stability of prodrugs at physiological pH
values is of the essence. In its absence, the prodrug will
(9) Benneche, T.; Strande, P.; Wiggen, U. Acta Chem. Scand. 1988,
43, 74-77.
(10) Felix, A.M.; Heimer, E.P.; Lambros, T.J .; Tzougraki, C.; Meien-
hofer, J . J . Org. Chem. 1978, 43, 4194-4196.
(11) Balajthy, Z.; Aradi, J .; Ildiko, T. K.; Elo¨di, P. J . Med. Chem.
1992, 35, 3344-3349.
to determine the rate of hydrolysis of the formaldehyde-
releasing 5-FU prodrugs: (a) direct measurement of the
absorbance decrease at 245 nm and (b) colorimetric
measurement of the formaldehyde concentration using

9086 J . Org. Chem., Vol. 62, No. 26, 1997
Menger and Rourk
Ta ble 1. Ra te Con sta n ts for Hyd r olysis of Ester in 10 a t
Va r iou s p H Va lu esa
corresponding to a half-life of 210 min. From a clinical
standpoint, this chemical stability is excellent.
kobs (min-1
)
t1/2 (min)
The large reactivity difference between 1a and 1b led
us to suspect that cleavage of 5-FU in 1a accompanied
by formaldehyde production was subject to intramolecu-
lar catalysis by the neighboring amide functionality.
Apparently, the amide of 1b is too distant to effect any
anchimeric assistance. If this is true, then an ester-
containing analog of 10, intermediate 5a , should be
relatively inert, and indeed such was found to be the case.
No hydrolysis was observed with 5a at pH 8.0 after 1 h
as judged by UV spectroscopy. We conclude, therefore,
that intramolecular catalysis in 1a and 10 by their amide
groups greatly accelerates the 5-FU ejection. Intramo-
lecular amide-catalyzed hydrolyses are well-known.13
Having disposed of the 5-FU end of things, we began
looking at the ara-C side. Again, release of the drug was
first examined with a model compound, 11. No activity
pH
4b
0.002
0.009
0.02
0.2
300
75
30
3
4.63b
5.0b
6.0c
a
5 × 10-5 M substrate, 25 ( 1 °C, as determined by decrease
in absorbance at 246 nm. 0.1 M sodium acetate buffer. c 0.05 M
b
potassium dihydrogen phosphate buffer.
Ta ble 2. Ra te Con sta n ts for Hyd r olysis of Ester in 1a a t
Va r iou s p H Va lu esa
pH
kobs (min-1)
t1/2 (min)
6.0b
6.5b
7.0b
7.4c
0.005
0.016
0.03
∼130
∼400
∼20
∼9
0.073
2 × 10-5 M substrate, 25 ( 1 °C, as determined by the
a
b
colorimetric method. 0.05 M potassium dihydrogen phosphate
buffer. c 0.05 m sodium dihydrogen phosphate buffer.
a modification of the Sawicki, et al. method.12 The
method is based on the condensation of formaldehyde
with 3-methyl-2-benzothiazolone hydrazone to form a
blue, cationic dye (eq 5), which can be determined
spectrophotometrically.
was observed in pH 7.0 or pH 8.0 buffers at 25 °C as
judged spectrophotometrically at 297 nm. Amide hy-
drolysis was forced upon the molecule only by subjecting
it to pH ) 10.0 buffer at 25 °C for several days; the
corresponding rate constant is 1.77 × 10-3 min-1 or a
half-life of about 400 min. Identical rate constants were
obtained for 1a and 1b. As expected and hoped for, ara-C
release via amide hydrolysis is inherently so slow that a
cellular enzyme is required to effect the reaction.
In summary, mutual prodrugs 1a and 1b were found
to have quite different reactivities. Prodrug 1a is labile
on its 5-FU side, but prodrug 1b is chemically stable at
the attachment sites of both 5-FU and ara-C. Biological
testing with the compounds is now underway.
Following the results of Bundgaard, we synthesized a
mutual prodrug analog with aniline replacing the ara-C
unit (10). Rate constants for hydrolysis were obtained
by monitoring the absorbance decrease at 246 nm in
acidic buffers ranging from pH 4.0 to 6.0. The data, given
in Table 1, give a linear log kobs vs pH profile with a slope
of unity. Above pH 6.0 the rates were too fast to measure
by conventional means.
Exp er im en ta l Section
P r ep a r a tion of Com p ou n d s 3. Gen er a l P r oced u r e.
Aliphatic diacid monoester 2 (33 mmol) and cesium carbonate
(33 mmol) were combined in 50 mL of DMF. The resultant
suspension was stirred for 30 min at room temperature and
then for 15 min in a 70 °C oil bath. After being cooled to room
temperature, the suspension was stirred in an ice bath for 30
min, and chloromethyl phenyl sulfide (30 mmol) was then
added via syringe. The mixture stirred for 3 h at room
temperature and at 70 °C for 15 min. The mixture was cooled
to room temperature, and 100 mL H2O was added. After 15
min of stirring, the suspended cesium carbonate went into
solution. The DMF/H2O mixture was then extracted with
diethyl ether (4 × 50 mL). The ether extracts were washed
with 1 M NaOH (1 × 50 mL) and brine (4 × 50 mL) and dried
over MgSO4. The ether was reduced in vacuo to give a clear
oil which was further purified (flash chromatography, 20%
ethyl acetate/petroleum ether) to afford the desired product
as a clear oil.
Cleavage of 5-FU from mutual prodrug 1a was followed
using the colorimetric assay for formaldehyde. The data
are summarized in Table 2. It is seen that the hydrolysis
rates are not as fast as with 10 (e.g. the half-lives at pH
6.0 for 1a and 10 being 130 and 3 min, respectively).
Nonetheless, the half-life for 1a at a physiological pH of
7.4 is about 9 min, which is dangerously close to render-
ing the compound clinically useless. Fortunately, 1b
proved to be inert from pH 4.0 to 7.4 in sharp contrast
to 1a . Even at a high pH of 10.0, the mutual prodrug
Su ccin ic a cid ben zyl ester (p h en ylsu lfa n yl)m eth yl
ester (3a ): 80% yield; IR (neat) 3460, 3058, 3032, 2951, 1738
cm-1 1H NMR (CDCl3) δ 2.69 (s, 4H), 5.12 (s, 2H), 5.41 (s,
;
2H), 7.29 (m, 10 H); 13C NMR (CDCL3) δ 29.00, 29.27, 66.61,
1b cleaves with a rate constant of only 3.3 × 10-3 min-1
,
68.41, 127.41, 128.24, 128.29, 128.57, 129.14, 130.40, 134.63,
(12) Sawicki, E.; Hauser, T. R.; Stanley, T. W.; Elbert, W. Anal.
Chem. 1961, 33, 93-96.
(13) Cohen, T.; Lipowitz, J . J . Am. Chem. Soc. 1961, 83, 4866-4877.

5-Fluorouracil/Cytarabine Mutual Prodrugs
J . Org. Chem., Vol. 62, No. 26, 1997 9087
135.72, 171.56, 171.83. Anal. Calcd for C18H18O4S (330.33):
C, 65.44; H, 5.49; S, 9.71. Found: C, 65.35; H, 5.51; S, 9.79.
Hexa n ed ioic a cid ben zyl ester (p h en ylsu lfa n yl)m eth yl
173.19, 173.54. Anal. Calcd for C18H19FN2O6 (378.36): C,
57.12; H, 5.06; F, 5.02; N, 7.41. Found: C, 57.19; H, 5.07; N,
7.35.
P r ep a r a tion of Com p ou n d s 6. Gen er a l P r oced u r e.
Into a 100 mL round bottom flask was added 5 (3 mmol) and
17 mL of acetic acid. Complete dissolution occurred after
about 30 min of stirring at rt. To the solution were added 10%
Pd/C (1.0 g) and 1,4-cyclohexadiene (15.8 mmol, 1.5 mL).
Periodic monitoring by TLC indicated that the reaction was
complete after 1 h. THF (25 mL) was added to the reaction
mixture (to make it less viscous and easier to filter). The
mixture was filtered first through a cotton plug and then
through a 0.45 µm nylon membrane filter to remove the Pd/C.
The THF was removed in vacuo, and then 50 mL of heptanes
was added. The acetic acid was removed by bulb-to-bulb
distillation as an acetic acid/heptanes azeotrope. The white
solid which remained after distillation was 6. 6 often con-
ester (3b): 94% yield; IR (neat) 3465, 3065, 3038, 2931, 1735
cm-1 1H NMR (CDCl3) δ 1.66 (m, 4H), 2.35 (t, J ) 6.6 Hz,
;
4H), 5.09 (s, 2H), 5.39 (s, 2H), 7.33 (m, 10H); 13C NMR (CDCl3)
δ 24.32, 24.36, 33.96, 34.04, 66.34, 68.09, 127.49, 128.34,
128.69, 129.24, 130.49, 134.80, 136.10, 172.66, 173.14. Anal.
Calcd for C20H22O4S (358.46): C, 67.02; H, 6.19; S, 8.93.
Found: C, 66.89; H, 6.26; S, 8.80.
P r ep a r a tion of Com p ou n d s 4. Gen er a l P r oced u r e.
Diester 3 (20 mmol) was dissolved in 40 mL of CH2Cl2 in a
three-neck round bottom flask equipped with an addition
funnel and purged with argon. Sulfuryl chloride (24 mmol,
24 mL of a 1.0 M CH2Cl2 solution) was added to the addition
funnel and slowly dropped into the reaction vessel. The
mixture changed color, from clear to dark orange, upon
addition of the sulfuryl chloride. After 1 h of stirring at room
temperature, crystalline cis-4-cyclohexene-1,2-dicarboxylic acid
(20 mmol) was added to the reaction mixture via powder
funnel. The color changed again, after about 30 min of
stirring, from dark orange back to the original clear. The
mixture was stirred another 30 min, and a white precipitate
formed. The precipitate was filtered and the solvent reduced
in vacuo to produce an oily residue. The residue was dissolved
in 50 mL of diethyl ether, and the ether solution was washed
with 10% NaHCO3 (1 × 50 mL) and H2O (2 × 50 mL), dried
over MgSO4, filtered, and reduced in vacuo to afford 4 as a
clear oil.
1
tained residual acetic acid by H NMR.
Su ccin ic a cid Mon o[(5-flu or o-2,4-d ioxo-3,4-d ih yd r o-
2H-p yr im id in -1-yl)m eth yl] ester (6a ): 99% yield; mp 153-
156 °C; IR (KBr) 3432, 3045, 2936, 2852, 1745, 1695, 1661 cm-1
1H NMR (CD3OD) δ 2.62 (s, 4H), 5.66 (s, 2H), 7.89 (d, J ) 6
Hz, 1H), (DMSO-d6) δ 2.32-2.61 (m, combined with solvent
signal, > 4H), 5.51 (s, 2H), 8.02 (d, J ) 6 Hz, 1 H), 11.99 (br,
1H); 13C NMR (DMSO-d6) δ 28.79, 28.66, 70.45, 129.50 (d, J CCF
) 34 Hz), 139.42 (d, J CF ) 229 Hz), 149.26, 157.45 (J CCF ) 25
Hz), 172.09, 173.33. Anal. Calcd for C9H9FN2O6 (260.18): C,
41.55; H, 3.49; F, 7.30; N, 10.77. Found: C, 41.35; H, 3.80; N,
10.07.
Hexa n ed ioic a cid m on o[(5-flu or o-2,4-d ioxo-3,4-d ih y-
d r o-2H-p yr im id in -1-yl)m eth yl] ester (6b): 60% yield; mp
171-173 °C; IR (KBr) 3430, 3138, 3038, 2951, 2838, 1751,
Su ccin ic a cid ben zyl ester ch lor om eth yl ester (4a ):
90% yield; IR (neat) 1765, 1735 cm-1; 1H NMR (CDCl3) δ 2.70
(s, 4H), 5.13 (s, 2H), 5.66 (s, 2H), 7.33 (s, 5H); 13C NMR (CDCl3)
δ 28.77, 28.97, 66.73, 68.77, 128.27, 128.34, 128.57, 135.62,
170.44, 171.64. Anal. Calcd for C12H13O4Cl (256.69): C, 55.24;
H, 5.15; Cl, 13.66. Found: C, 55.01; H, 5.12; Cl, 13.51.
1
1735, 1700, 1660 cm-1; H NMR (DMSO-d6) ( 1.49 (t, J ) 3.6
Hz, 4H), 2.18 (t, J ) 6.4 Hz, 2H), 2.34 (t, J ) 6.4 Hz, 2H), 5.55
(s, 2H), 8.11 (d, J ) 6.6 Hz, 1H), 11.00 (br, 1H); 13C NMR
(DMSO-d6) δ 23.64, 23.78, 32.84, 33.25, 70.49, 129.43 (d, J CCF
) 34 Hz), 139.42 (d, J ) 230 Hz), 149.23, 157.40 (d, J CCF ) 26
Hz), 172.45, 174.24. Anal. Calcd for C11H13FN2O6 (288.23):
C, 45.82; H, 4.55; F, 6.59; N, 9.51. Found: C, 46.05; H, 4.66;
N, 9.51.
H exa n ed ioic a cid b en zyl est er ch lor om et h yl est er
1
(4b): 98% yield; IR (neat) 1762, 1735 cm-1; H NMR (CDCl3)
δ 1.69 (m, 4H), 2.39 (m, 4H), 5.12 (s, 2H), 5.69 (s, 2H), 7.35 (s,
5H); 13C NMR (CDCl3) δ 24.07, 24.29, 33.72, 33.93, 66.40,
68.75, 128.30, 128.38, 128.72, 136.10, 171.39, 173.10. Anal.
Calcd for C14H17ClO4 (284.74): C, 59.14; H, 6.03; Cl, 12.31.
Found: C, 59.03; H, 5.99; Cl, 12.48.
P r ep a r a tion of Com p ou n d s 7. Gen er a l P r oced u r e. In
a 100 mL round bottom flask was dissolved 6 (2mmol) in 5
mL of DMF. Pentafluorophenol (2 mmol) was added followed
by EDCI‚HCl (2 mmol). The mixture stirred at room temper-
ature for 2 h. The DMF was removed by bulb-to-bulb distil-
lation, and the resulting residue was purified (flash chroma-
tography, 40% EtOAc/CH2Cl2, solid deposition on silica gel)
to give 7. 7 must be used immediately as it decomposes on
standing.
Su ccin ic a cid 5-(flu or o-2,4-d ioxo-3,4-d ih yd r o-2H -
p yr im id in -1-yl)m eth yl ester 2,3,4,5,6-p en ta flu or op h en yl
ester (7a ): 43% yield; mp 130-131 °C; 1H NMR (CDCl3) δ 2.85
(m, 2H), 3.04 (m, 2H), 5.69 (s, 2H), 7.59 (d, J ) 5 Hz, 1H),
8.76 (br, 1H).
P r ep a r a tion of Com p ou n d s 5. Gen er a l P r oced u r e.
Into a 100 mL, three-neck flask equipped with stirring bar
and addition funnel was placed 5-fluorouracil (8.11 mmol) and
10 mL of DMF. The mixture stirred for 10 min, and then a
3-fold excess of triethylamine (24.33 mmol) was added via
syringe. The mixture stirred at room temperature for 30 min.
Freshly prepared (less than 24 h old) 4 (8.11 mmol), dissolved
in 10 mL of DMF, was added dropwise via addition funnel. A
precipitate of triethylamine hydrochloride formed after about
1 h of stirring. The stirring continued overnight (16 h), and
then the precipitate was filtered and the DMF removed by
bulb-to-bulb distillation. The resultant crude solid gave 5 as
a white solid after purification by flash chromatography (40%
EtOAc/CH2Cl2, solid deposition on silica gel with THF).
Hexa n ed ioic a cid 5-(flu or o-2,4-d ioxo-3,4-d ih yd r o-2H-
p yr im id in -1-yl)m eth yl ester 2,3,4,5,6-p en ta flu or op h en yl
ester (7b): 77% yield; mp 118 °C; 1H NMR (CDCl3) δ 1.79 (m,
4H), 2.47 (t, J ) 6.9 Hz, 2H), 2.70 (t, J ) 6.9 Hz, 2H), 5.66 (s,
2H), 7.62 (d, J ) 5 Hz, 1H), 9.39 (br, 1H).
Su ccin ic a cid ben zyl ester (5-flu or o-2,4-d ioxo-3,4-d i-
h yd r o-2H-p yr im id in -1-yl)m eth yl ester (5a ): 76% yield; mp
125-127 °C; IR (KBr) 3432, 3178, 3072, 2832, 1758, 1711, 1658
N-(1-â-D-Ar a bin ofu r a n osylcytosyl)su ccin a m ic Acid (5-
F lu or o-2,4-d ioxo-3,4-d ih yd r o-2H-p yr im id in -1-yl)m eth yl
Ester (1a ). Ara-C (0.15 g, 0.62 mmol) and activated ester 7a
(0.46 g, 1.08 mmol) were combined in 2 mL of DMF. The
mixure stirred at room temperature for 72 h. The DMF was
removed by bulb-to-bulb distillation, and a clear viscous
residue remained. The residue was dissolved in a minimum
(2-3 mL) of methanol and then rapidly precipitated out of
solution by addition of 500 mL of CHCl3 (it is critical that
contact with methanol be kept to a minimum). The precipitate
was then immediately filtered through a 0.45 µm nylon
membrane and dried in a vacumn dessicator at 60 °C. In order
to remove persistent CHCl3 after drying, the precipitate was
dissolved in 2-3 mL of deionized H2O, rapidly frozen solid,
and lyophilized. This process was repeated twice to afford 284
mg of analytically pure 1a (93%): mp 126 °C dec; IR (KBr)
3412 (br), 3078, 2931, 1721, 1654, 1565, 1491, 1391, 1314,
cm-1 1H NMR (CDCl3) δ 2.71 (s, 4H), 5.12 (s, 2H), 5.63 (s,
;
2H), 7.34 (s, 5H), 7.56 (d, J ) 5 Hz, 1H), 9.45 (br, 1H); 13C
NMR (CDCl3) δ 28.42, 28.44, 65.67, 70.55, 127.87, 128.00,
128.40, 129.47 (d, J CCF ) 34 Hz), 135.9, 139.35 (d, J CF ) 229
Hz), 149.19, 157.38 (d, J CCF ) 26 Hz), 171.66, 171.72; 19F NMR
(qualitative, no internal standard, CDCl3) δ 9.93 (d, J FH ) 5
Hz). Anal. Calcd for C16H15FN2O6 (350.30): C, 54.84; H, 4.32;
F, 5.42; N, 8.00. Found: C, 55.05; H, 4.39; N, 7.82.
Hexa n ed ioic a cid ben zyl ester (5-flu or o-2,4-d ioxo-3,4-
d ih yd r o-2H-p yr im id in -1-yl)m eth yl ester (5b): 63% yield;
mp 98 °C; IR (KBr) 3405, 3172, 3038, 2838, 1751, 1711, 1658
cm-1; 1H NMR (CDCl3) δ 1.67 (m, 4H), 2.36 (q, J ) 7 Hz, 4H),
5.11 (s, 2H), 5.63 (s, 2H), 7.35 (s, 5H), 7.62 (d, J ) 5 Hz, 1H),
9.73 (br, 1H): 13C NMR (CDCl3) δ 24.06, 24.24, 33.54, 33.85,
66.46, 69.86, 128.38, 128.40, 128.70 (d, J ) 34 Hz), 128.73,
136.07, 140.36 (d, J ) 239 Hz), 149.58, 157.22 (d, J ) 27 Hz),

9088 J . Org. Chem., Vol. 62, No. 26, 1997
Menger and Rourk
1131, 1050 cm-1; 1H NMR (DMSO-d6) δ 2.61 (t, J ) 5 Hz, 2H),
2.69 (t, J ) 5 Hz, 2H), 3.61 (d, J ) 4 Hz, 2H), 3.82 (s, 1H),
3.91 (s, 1H), 4.05 (s, 1H), 5.10 (br, 1H), 5.47 (br, 2H), 5.56 (s,
2H), 6.04 (d, J ) 4 Hz, 1H), 7.12 ( d, J ) 4 Hz, 1H), 8.03 (d, J
) 7 Hz, 1H), 8.08 (d, J ) 6 Hz, 1H), 10.92 (s, 1H), 11.90 (br,
1H); 13C NMR (DMSO-d6) δ 27.98, 31.05, 61.07, 71.83, 74.64,
76.20, 85.80, 87.05, 94.29, 129.51 (d, J CCF ) 34 Hz), 139.42
(J CF ) 229 Hz), 146.73, 149.30, 154.51, 157.50 (d, J CCF ) 25
Hz), 162.05, 172.05, 172.41; 19F NMR (qualitative, no internal
standard, DMSO-d6) δ 9.93 (d, J FH ) 5 Hz). Anal. Calcd for
H18H20FN5O10‚H2O (503.40): C, 42.95; H, 4.41; F, 3.77; N,
13.91. Found: C, 42.84; H, 4.38; N, 13.92.
Kin etics. Ultraviolet and visible spectral measurements
were performed with a Varian DMS 200 spectrophotometer
or a Varian DMS 300 spectrophotometer. Measurement of pH
was carried out with a Corning pH meter 130 at the temper-
ature of study. Aqueous buffers were either purchased from
Fisher Scientific or made in our laboratory. Buffers were made
in our laboratory by adjustment of a 0.1 M sodium acetate
solution (pH ) 3.7-5.6), 0.05 M sodium dihydrogen phosphate
solution (pH ) 6-8), or 0.025 M sodium carbonate/sodium
bicarbonate solution (pH ) 10) to the desired pH with 1 M
NaOH. UV method. The UV method is defined as recording
the absorbance changes over time for reactions in which the
absorbance of substrate and product differ maximally at a
particular wavelength. This method was used to study ara-C
activation of 1a , 1b, and model 11, as well as 5-FU activation
of model 10. Reactions were performed in 3 mL aliquots of
buffer solution in a temperature-controlled quartz cuvette after
initiation by addition of 50 µL of a substrate stock solution in
acetonitrile. Reaction rates were determined for hydrolysis
of the ara-C amide for both the mutual prodrugs (1a ,b) and
the model (11) by recording the the decrease in absorbance at
297 nm. Reaction rates were determined for 5-FU model 10
by recording the decrease in absorbance at 246 nm. Initial
concentration of all substrates studied by the UV method was
5 × 10-5 M. Psuedo-first-order rate constants were calculated
from plots of ln (Abst - Abs∞) vs time, where Abs∞ and Abst
are the absorbance at infinity and at time, t, respectively. A
pH measurement of the reaction buffer solution was taken
after each reaction had reached completion, and no change in
pH was observed.
Color im etr ic Meth od . 5-FU activation of 1a , 1b, and
model 5a was studied using this method. Formaldehyde
released by activation of the 5-FU side of the mutual prodrug
or model was determined using a modification8c of a colori-
metric method described by Sawicki et al.12 A sample (∼15
mg) of the solid prodrug or model was weighed out on an
analytical balance and added to 25 mL of buffer in a temper-
ature-controlled, water-jacketed, covered beaker equipped with
a stirring bar. All reactions were run at 25 ( 1 °C except for
5a , which was run at 37 ( 1 °C (physiological temperature).
The sample dissolved in less than 5 s to give a solution of ∼1.2
× 10-3 M substrate. At appropriate intervals, 1 mL samples
were withdrawn and diluted to 10 mL with water. A 500 µL
aliquot of the diluted test solution (∼1.2 × 10-4 M substrate)
was mixed with 400 µL of a 0.1 M acetate buffer (pH ) 4.0)
and 100 µL of a 0.5% aqueous solution of 3-methyl-2-ben-
zothiazolone hydrazone hydrochloride hydrate. After the
mixture was allowed to stand for 25-30 min at room temper-
ature (20-25 °C), 500 µL of a 0.25% aqueous iron(III) chloride
hexahydrate solution was added. After 10 min, 1500 µL of
water was added, and the absorbance of the mixture (∼2 ×
10-5 M substrate) was read vs a reagent blank at 625 nm. The
concentration of formaldehyde in the solution was calculated
by referring to a standard curve. The formaldehyde standard
curve was constructed using the aforementioned colorimetric
method to analyze seven aqueous formaldehyde solutions in
the range (2-20) × 10-6 M. The seven aqueous formaldehyde
solutions were made from either solid paraformaldehyde or a
37% aqueous formaldehyde solution analyzed for analytical
use by the gravimetric method of Yoe and Reid.14 The molar
absorptivity was found to be 4.44 × 104 L/mol‚cm. Pseudo-
first-order rate constants were calculated from the slopes of
linear plots of ln (Abs∞ - Abst) vs time, where Abs∞ and Abst
are the absorbance at infinity and at time, t, respectively.
Extent of reaction at infinity was determined using the
standard curve for formaldehyde. A pH measurement of the
buffer solution was taken after each reaction had reached
completion and no pH change was observed.
5-[(1-â-D -Ar a b i n o fu r a n o s y lc y t o s y l)c a r b a m o y l]-
p en ta n oic Acid (5-F lu or o-2,4-d ioxo-3,4-d ih yd r o-2H-p y-
r im id in -1-yl)m eth yl Ester (1b). Activated ester 7b (0.44,
200 mg) and ara-C (0.44 mmol, 107 mg) were combined in 5
mL of DMF and stirred at room temperature. The reaction
was monitored periodically by TLC (1:4 MeOH/CHCl3). After
4 days, the DMF was removed by bulb-to-bulb distillation, and
the residue was purifed (flash chromatograhy, solid deposition
on silica with 1:1 THF/CH3CN plus a few drops MeOH,
gradient elution, 1:1 THF/CHCl3 followed by 100% THF) to
give 150 mg of 1b (66% yield): mp 105 °C dec; IR (KBr) 3430,
3078, 2945, 1720, 1665, 1645, 1565, 1490, 1388, 1314, 1124
1
cm-1; H NMR (DMSO-d6) δ 1.54 (m, 4H), 2.37 (m, 4H), 3.62
(m, 2H), 3.83 (m, 1H), 3.92 (m, 1H), 4.06 (m, 1H), 5.07 (br,
1H), 5.49 (d, J ) 4.8 Hz, 2H), 5.63 (s, 2H), 6.05 (d, J ) 3.9 Hz,
1H), 7.22 (d, J ) 3 Hz, 1H), 8.05 (d, J ) 7.5 Hz, 1H), 8.13 (d,
J ) 6.6 Hz, 1H), 10.84 (s, 1H), 11.97 (br, 1H); 13C NMR (DMSO-
d6) δ 23.59, 23.79, 32.84, 35.94, 61.05, 70.53, 74.63, 76.15,
85.79, 87.02, 94.32, 129.49 (d, J ) 34 Hz), 139.47 (d, J ) 229
Hz), 146.73, 149.27, 154.52, 157.44 (d, J ) 26 Hz), 162.17,
172.48, 173.58. Anal. Calcd for C20H24FN5O10‚H2O (531.46):
C, 45.20; H, 4.93; F, 3.57; N, 13.18. Found: C, 45.32; H, 4.88;
N, 12.99.
N-P h en ylsu ccin a m ic Acid (5-F lu or o-2,4-d ioxo-3,4-d i-
h yd r o-2H-p yr im id in -1-yl)m eth yl Ester (10). Succinate
half-acid 6a (0.26 g, 1 mmol) was dissolved in 5 mL of DMF.
Pentafluorophenol (0.184 g, 1 mmol) was added and allowed
to dissolve, followed by EDCI‚HCl (0.19 g, 1 mmol). The
mixture stirred at room temperature for 2 h. Aniline (0.93 g,
1 mmol) was then added and the mixture stirred for ∼16 h.
The DMF was removed by bulb-to bulb distillation and the
residue purified (flash chromatography, two separate columns,
first eluant 90% EtOAc/hexanes, second eluant 1:1 acetone/
chloroform) to give 200 mg of 10 (59% yield): mp 155 °C; IR
(KBr) 3138, 3080, 2838, 1728, 1658,1257 cm-1
;
1H NMR
(DMSO-d6) δ 2.62 (s, 4H), 5.58 (s, 2H), 7.00 (t, J ) 8 Hz, 1H),
7.26 (t, J ) 8 Hz, 2H), 7.54 (d, J ) 8 Hz, 2H), 8.08 (d, J ) 7
Hz, 1H), 9.98 (s, 1H), 11.97 (s, 1H); 13C NMR (DMSO-d6) δ
28.53, 30.64, 70.43, 118.90 122.97, 128.64, 129.48 (d, J CCF
)
34 Hz), 139.13, 139.37 (d, J CF ) 26 Hz), 149.22, 157.38 (d, J CCF
) 26 Hz) 169.56, 172.19.
N-(1-â-D-Ar abin ofu r an osylcytosyl)su ccin am ic Acid Ben -
zyl Ester (11). 2a (1.04 g, 5 mmol) was dissolved in 10 mL
of DMF. EDCI‚HCl (0.96 g, 5 mmol) and pentafluorophenol
(0.58 g, 5 mmol) were added, and the mixture was stirred
overnight. The intermediate succinic acid benzyl ester 2,3,4,5,6-
pentafluorophenyl ester was separated from the reaction
mixture (silica gel plug, 25%EtOAc/petroleum ether), and a
portion (0.61 g, 2 mmol) dissolved in 5 mL of DMF. Ara-C
(0.243 g, 1 mmol) was added, and the mixture stirred was
overnight. The DMF was removed in vacuo and the residue
purified (flash chromatography, 3:1 tetrahydrofuran/chloro-
form) to afford 0.33 g of 11 (76% yield): mp 88-90 °C; IR (KBr)
1
3330 (br), 1724, 1644 cm-1; H NMR (DMSO-d6) δ 2.67 (dt, J
) 3, 13 Hz, 4H), 3.59 (dd, J ) 5, 7 Hz, 2H), 3.81 (dd, J ) 3, 5
Hz, 1H), 3.92 (d, J ) 3 Hz, 1H), 4.04 (d, J ) 5 Hz, 1H), 5.06 (s,
3H), 5.50 (t, J ) 4 Hz, 2H), 6.05 (d, J ) 4 Hz, 1H), 7.15 (d, J
) 8 Hz, 1H), 7.33 (s, 5H), 8.05 (d, J ) 8 Hz, 1H), 10.92 (s, 1H);
13C NMR (DMSO-d6) δ 29.39, 32.49, 62.55, 67.21 76.27, 77.87,
87.20, 89.16, 96.72, 128.97, 129.04, 129.41, 137.38, 147.70,
157.49, 163.79, 173.61, 173.94. Anal. Calcd for C20H23N3O8
(433.42): C, 54.52; H, 5.35; N, 9.69. Found: C, 54.31; H, 5.44;
N, 9.47.
Ack n ow led gm en t. This work was supported by the
National Institutes of Health.
J O971076P
(14) Yoe, J . H.; Reid, L. C. Ind. Eng. Chem., Anal. Ed. 1941, 13,
238-240.